Proc. Nati. Acad. Sci. USA Vol. 89, pp. 1320-1324, February 1992 Immunology

Cytotoxic and viral neutralizing antibodies crossreact with streptococcal M protein, enteroviruses, and human cardiac myosin (molecular mimicry/autoimmunity)

MADELEINE W. CUNNINGHAM*t, SUSAN M. ANTONE*, JAMES M. GULIZIAt, BRUCE M. MCMANUSt, VINCENT A. FISCHETTI§, AND CHARLES J. GAUNTrT *Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190; lUniversity of Texas Health Science Center, San Antonio, TX 78284-7758; tDepartment of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198; and §Laboratory of Bacteriology and Immunology, The Rockefeller University, New York, NY 10021-6399

Communicated by Maclyn McCarty, November 4, 1991 (received for review September 26, 1991)

The streptococcal M protein has been conclusively shown to crossreact immunologically with myosin (9, 21, 22, 24-26) and tropomyosin (22), and myosin-specific antibodies affinity-purified from sera of patients with acute rheumatic fever have been shown to crossreact with M protein (9, 26). In fact, a pentameric amino acid sequence (Gln-Lys-Ser-Lys-Gln) in M protein was involved in the crossreaction of anti-myosin antibodies from acute rheumatic fever patients with synthetic peptides representing the M protein molecule (26). Murine and human mAbs that were crossreactive with streptococcal M protein and myosin were also found to recognize synthetic peptides that contained the same pentameric sequence (26). The a-helical coiled-coil structure of the streptococcal M protein (27-29) provides a basis for immunological crossreactivity with myosin (9, 21, 22, 24-26). Little is known about the conformation of the epitopes of infectious agents that are associated with induction of autoantibodies. The hypothesis that different types of infectious agents such as viruses and bacteria share similar or common structures has not been established. Groups or families of autoantigenic structures may be important in generating autoimmune responses in genetically predisposed individuals with deleterious consequences. Although it is clear that autoantibodies to myosin, actin, and many other host proteins can be present in individuals without serious consequences (2, 30, 31), antibodies crossreactive with host cell surface epitopes may be cytotoxic and cause tissue damage (32). Because viruses and group A streptococci have been implicated in autoimmune diseases of the heart, we tested the hypothesis that viruses, such as group B CVs, might share epitopes with the group A streptococcus. Streptococcal M protein and viral capsid proteins are surface targets for antibody and complement-mediated clearance (33-35). To test this hypothesis, a panel of murine IgM mAbs reactive with group A streptococcal M protein and heart tissue antigen was used in this study. These antibodies have been extensively characterized (17, 18, 21, 22, 26, 32) and shown to be broadly crossreactive with a-helical coiled-coil protein structures including myosin (9, 17, 18), tropomyosin (22), keratin (21), vimentin (36), and laminin (32). We now present evidence that this crossreactivity between a-helical structures gives these anti-streptococcal mAbs dual specificity for both bacterial and viral pathogens. We show that these mAbs recognize specific viral capsid proteins and neutralize virus that is intimately associated with the production of autoimmune heart disease in mice (10, 11) and humans (11-14).

The development of autoimmunit in certain ABSTRACT instances is related to infectious agents. In this report, cytotoxic monoclonal antibodies (mAbs) that recognize epitopes on both enteroviruses and the bacterium Streptococcus pyogenes are described. Murine anti-streptococcal mAbs that were crossreactive with streptococcal M protein, human cardiac myosin, and other a-helical coiled-coil molecules were found to neutralize coxsackieviruses B3 and B4 or poliovirus type 1. The viral-neutralizing anti-streptococcal mAbs were also cytotoxic for heart and fibroblast cell lines and reacted with viral capsid proteins on a Western Immunoblot. Alent of amino acid sequences shared between streptococcal M protein, coxsackievirus B3 capsid protein VP1, and myosin revealed 40% identity in a 14- to 15-amino acid overlap. Synthetic peptides containing these sequences blocked mAb reactivity with streptococcal M protein. The data show that antibodies against a-helical structures of bacterial and viral antigens can lead to cytotoxic reactions and may be one mechanism to explain the origin of autoimmune heart disease.

Viruses as well as other microbial agents have been associated with autoimmune diseases (1-6). In rheumatic carditis (7-9) and myocarditis (10-16), substantial evidence has connected a bacterial or viral infection directly with autoimmunity. In many autoimmune diseases, the connection with infectious agents is not clear; however, molecular mimicry may play a role in deregulating the immune system in susceptible hosts (1-6, 9-16). In rheumatic carditis, antibodies to heart tissue were shown to react with the group A streptococcus, the etiologic agent of acute pharyngitis and acute rheumatic fever (8). Heart-reactive antibodies were also found in sera of patients with myocarditis, a disease that often results from a coxsackievirus (CV) group B infection (10-14), and in sera of mice with CVB3-induced myocarditis (15, 16). Antibodies to heart tissue in both diseases recognized myosin as a major autoantigen (9, 17-20). Studies of murine and human monoclonal antibodies (mAbs) directed against the group A streptococcus and myosin revealed two distinct groups of heartreactive antibodies (9, 21, 22). One group recognized a-helical coiled-coil molecules such as myosin, keratin, and tropomyosin and included antibodies recognizing actin, whereas the other group recognized DNA and myosin (21, 22). A recent report (23), unrelated to the streptococcal studies, identified a mAb that crossreacted with the capsid protein VP1 of CV serotype B4 and with myosin. Although there are many studies, the role of immunological crossreactivity in disease has not been established.

MATERIALS AND METHODS Preparation of Virus. Viruses were prepared as described (37) and were purified by ultracentrifugation (38). Purified

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Abbreviations: mAb, monoclonal antibody; HCMB, human cardiac myosin chain; CV, coxsackievirus. tTo whom reprint requests should be addressed.

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Immunology: Cunningham et al. virus particles were dissolved in 1% SDS and viral polypeptides were separated by SDS/PAGE (21). Separated proteins were blotted onto nitrocellulose and incubated with antibody culture fluids as described (21). mAbs. Murine mAbs were produced, maintained, and stored as described (18, 21). Antigen specificities of IgM mAbs 36.2.2,49.8.9, 54.2.8, 101.4.1,24.1.2, and 654.1.1 have been described (17, 18,21,22,26,32,36). Briefly, mAb 36.2.2 has been shown to react with group A streptococcal M proteins serotypes 1, 5, and 6 and with the following host proteins: myosin, tropomyosin, actin, keratin, and laminin. mAb 49.8.9 has been shown to react with streptococcal M5 and M6 proteins and with the host protein vimentin (36). mAb 54.2.8 reacts with streptococcal M5 and M6 proteins and with the host proteins myosin and tropomyosin as well as with DNA (21, 22). mAbs 101.4.1, 654.1.1, and 24.1.2 have also been shown to react with various streptococcal M proteins and myosin (22). ELISA. Virus antigen was applied to microtiter plates at 50 ng/ml. M6 protein was applied to microtiter plates at 10 ,ug/ml. The ELISA was performed as described (17, 18). Results were calculated from triplicate measurements. Plaque-Reduction Assay. Neutralization of CVB3 or CVB4 variants and poliovirus type 1 by anti-streptococcal mAbs 36.2.2, 49.8.9, and 54.2.8 was tested in plaque-reduction assays. A known number of virus plaque-forming units were mixed 1:1 (vol/vol) with mAb or culture medium control. Antibodies or controls were partially purified by 50%o ammonium sulfate saturation and precipitation. IgM concentrations of purified mAb preparations were tested by ELISA (9). The mAb/virus mixture was incubated 45 min at 37°C, 0.1 ml was plated in duplicate on HeLa cell monolayer cultures, and a standard plaque assay was performed (37). The average number of plaques in the virus controls without antibody ranged between 80 and 352 for the CVB3 variants, 79 and 146 for CVB4, 40 and 86 for poliovirus type 1, and 30 and 117 for CVB3m. Competitive Inhibition ELISA. Competitive inhibition of mAb binding to streptococcal M6 protein was tested using synthetic peptides. Percent inhibition was determined after incubating the mAb with peptide (500 ,ug/ml), 1:1 molar ratio, as described (18, 26), and the mixture was then incubated with the M6 protein in the ELISA. Results were calculated from triplicate measurements. Peptides contained the following sequences: M6 peptide was Lys-Leu-Thr-Glu-Lys-GluLys-Ala-Glu-Leu-Gln-Ala-Lys-Leu-Glu-Ala-Glu, beginning at residue 326 of the mature streptococcal M protein (39); CVB3 peptide was Tyr-Ala-Glu-Trp-Val-Leu-Thr-Pro-Arg-

Gln-Ala-Ala-Gln-Leu-Arg-Arg-Lys-Leu-Glu-Phe-Phe, beginning at residue 87 of the CV VP1 (40); human cardiac myosin, p-chain (HCMB) peptide was Glu-Ala-Glu-Ala-SerLeu-Glu-His-Glu-Glu-Gly-Lys-Ile-Leu-Arg-Ala-Gln-LeuGlu-Phe-Asn, beginning at residue 1544 of the myosin heavy chain (55). Cell Lines and Cytotoxicity Assay. Cell lines were obtained from the American Type Culture Collection as a primary rat heart cell line (ATCC CRL-1446), a primary rat fibroblast cell line (ATCC CRL-1213), or a primary rat liver cell line (ATCC CRL-1439). Cells were cultured overnight in sterile 96-well microtiter plates at 1 x 104 cells per well at 37°C and 5% C02/95% air. The cells were labeled with Na251CrO4 (DuPont) at 5 ,uCi per well (1 Ci = 37 GBq) for 2 h at 37°C. Culture medium was removed and the attached cells were washed three times with culture medium containing 20% (vol/vol) fetal bovine serum and incubated for 1 h prior to addition of antibody. Antibody was added to the cells at 50 ,ul per well for 1 h at 37°C. An equal volume of guinea pig complement (Whittaker Bioproducts) was added and the mixture was incubated for 1 h at 37TC. Supernatant fluids were harvested by a Skatron harvester system and 51Cr release was measured

Proc. Natl. Acad. Sci. USA 89 (1992)

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in a LKB y-counter (LKB-1282 Compugamma). Minimum lysis was calculated from cells treated with culture medium alone and 100% lysis was calculated from cells treated with 1 M HCl for maximum 51Cr release. Percent lysis was determined by calculating [(test sample release - minimum release)/(maximum release - minimum release)] x 100.

RESULTS Reaction of Virus with Anti-Streptococcal mAbs. In the ELISA (Table 1), anti-streptococcal IgM mAbs 36.2.2, 49.8.9, and 54.2.8 were incubated with purified CVB3m particles, a variant highly myocarditic for mice (38). The mAbs that recognize (17, 18, 21, 26) shared epitopes on streptococcal M protein and cardiac myosin also recognized epitopes on the partially denatured CV particles. A mouse anti-CVB3m serum reacted strongly with the virus particles in the ELISA (Table 1). Mouse IgM, a medium control, and two other IgM anti-streptococcal mAbs (101.4.1 and 24.1.2) did not react with the virus in this assay. Electrophoretically separated capsid proteins from purified CVB3m and from poliovirus type 1 were incubated with anti-streptococcal mAbs 36.2.2, 49.8.9, or 54.2.8 on Western blots (Fig. 1). All three anti-streptococcal mAb probes uniquely recognized viral proteins of CVB3m but did not react with proteins of poliovirus type 1. mAb 36.2.2 reacted most strongly with VP3 of CVB3m, whereas mAb 54.2.8 reacted exclusively with VP1 of CVB3m. mAb 49.8.9 recognized an epitope in VP1, VP2, and VP3 of CVB3m. The reaction of mAb 49.8.9 with more than one capsid protein may be surprising; however, recent data show (41) that a mAb against enterovirus 70 neutralized the virus and reacted with two viral capsid proteins. Nevertheless, our data clearly document epitope sharing in group A streptococcal M protein, cardiac myosin, and the CVB3m capsid proteins. Neutralization of Virus by Anti-Streptococcal mAbs. After establishing that anti-streptococcal mAbs reacted with CVB3m capsid proteins, the mAbs were tested for virus neutralization in a plaque-reduction assay (37). CVs (one CVB4 variant and eight CVB3 variants) and poliovirus type 1 were incubated with partially purified mAbs 36.2.2, 49.8.9, and 54.2.8 (Table 2). By using 50% reduction in plaque number as the endpoint, mAbs 36.2.2 and 54.2.8 neutralized poliovirus type 1, whereas CVB4 and the other CVB3 variants were not consistently or significantly (.50%) neutralized. Conversely, mAb 49.8.9 neutralized seven of eight CVB3 variants and a CVB4 variant but did not consistently or significantly neutralize poliovirus type 1. Cross neutralization of viral serotypes was recently shown to occur when an anti-poliovirus type 2 mAb neutralized poliovirus type 1 (42). The anti-streptococcal mAbs possessed various neutralizing capabilities with significant virus neutralization concentrations ranging from 86 ,ug of IgM per ml for mAb 49.8.9 to 1.999 mAb 49.8.9 1.805 mAb 54.2.8 1.999 mAb 101.4.1 0.191 mAb 24.1.2 0.070 Anti-CVB3 serum >1.999 Medium control 0.004 Mouse IgM (20 Ag/ml) 0.080 IgM concentrations were